Decoding molecular programs in melanoma brain metastases

Melanoma brain metastases (MBM) variably respond to therapeutic interventions; thus determining patient’s prognosis. However, the mechanisms that govern therapy response are poorly understood. Here, we use a multi-OMICS approach and targeted sequencing (TargetSeq) to unravel the programs that potentially control the development of progressive intracranial disease. Molecularly, the expression of E-cadherin (Ecad) or NGFR, the BRAF mutation state and level of immune cell infiltration subdivides tumors into proliferative/pigmented and invasive/stem-like/therapy-resistant irrespective of the intracranial location. The analysis of MAPK inhibitor-naive and refractory MBM reveals switching from Ecad-associated into NGFR-associated programs during progression. NGFR-associated programs control cell migration and proliferation via downstream transcription factors such as SOX4. Moreover, global methylome profiling uncovers 46 differentially methylated regions that discriminate BRAFmut and wildtype MBM. In summary, we propose that the expression of Ecad and NGFR sub- classifies MBM and suggest that the Ecad-to-NGFR phenotype switch is a rate-limiting process which potentially indicates drug-response and intracranial progression states in melanoma patients.

Whole transcriptome and methylome data were deposited in the European Genome-Phenome Archive (EGA), under accession numbers EGAS00001005976 and EGAS00001005975 (https://ega-archive.org/). The data are available under controlled access. Access can be obtained by contacting the appropriate Data Access Committee listed in the study. Access will be granted to commercial and non-commercial parties according to patient consent forms and data transfer agreements. A response to requests for data access can be expected within 14 days. After access has been granted, the data is available for two years. Supplementary tables have been deposited in Zenodo (https://zenodo.org/record/7013097). Kaplan-Meier survival data were derived from study EGAS00001003672 shown in Figure  Live cell imaging-based raw measurement files of drug response and proliferation assays are given in the Source Data file with this paper. The remaining data are available within the article, Supplementary Information or Source Data file.
We included all individuals (starting in 2017) with MBM where sufficient material was available as specified in the description of study design.
As whole transcriptome data of therapeutically (BRAFi, MEKi, ICi) treated MBM are not avaliable, we performed additional analyses on the following data sets: GSE77940 (pre-and post (BRAFi/MEKi) treatment melanoma (n= 6 patients; one was excluded from analysis). In addition Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. we performed analysis on the TCGA melanoma set (SKCM) containing 472 specimens (primary melanoma, n= 151; extracranial metastases, n=315 and brain metastases, n=6) and on expression data of FFPE samples of melanoma patiens (n=138) including intracranial (n= 79, BM) and extracranial metastases (n=59, EM) of study EGAS0000100367279 performed by Michael Davies.
No data were excluded from analyses.
Histological diagnosis of melanoma samples was performed by at least two consultants of (neuro)pathology with agreement. Histological stainings were replicated at least once with the appropriate positive and negative controls. Each replication was successful. Immunohistochemistry/-fluorescent analyses were technically replicated at least once. Each replication was successful.
All cell-based in vitro experiments were performed in six to eight technical and three biological replicates. Each replication was successful. RT-qPCR was performed in two to three technical and three biological replicates. Each replication was successful. The reliability of TargetSeq was demonstrated by comparative analyses of concordant sets of MBM and/or associated sets of MBM and BMCs. In addition, overlapping analyses of two indendent amplicon-panels and prior results from routine diagnostic (at least BRAF status) successfully confirmed the reliability of DNA sequencing. Knockdown of NGFR was additionally validated by immunoblotting which was replicated in five cell lines. Each replication was successful.
Randomization is not relevant for this study as we included all patients with MBM where sufficient material was available as described in the study design.
Blinding was implemented through use of unsupervised analysis. Targeted deep-DNA ssequencing, RNA-sequencing, real-time PCR, immunohistological stainings, IF stainings, and FACS analysis were performed in a blinded fashion.
The following antibodies were used in our study: Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.
All antibodies were checked for reproducability and integrity of the assay three times in independent staining experiments and in at least three different positive samples and compared to expected staining patterns in the controls regarding published expression of the antigens were applicable. Furthermore an additional secondary-only antibody control (i.e. omission of first antibody) was performed for every setup.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots. After removal of medium, cells were washed with PBS and harvested by Trypsin (0.05 % 206 Trypsin/EDTA). Following addition of cell culture medium, cells were collected by centrifugation at 330g at room temperature for 3 (min) and resuspended in 100 #l of ice cold buffer (PBS/0.5 % bovine serum/2 mM EDTA) and stored on ice. Cells were incubated with fluorescently labeled primary antibodies against CD271-PE (Miltenyi) DECMA1-APC (recognizing the N-terminal domain of Ecadherin, Biolegend), and non-labeled antibodies against AXL (Novus biologicals), PD-L1 (BioLegend), c-MET (MET, Cell signaling) or KBA.62 (BioLegend) diluted in buffer according to the manufacturer's specifications and stored at 4°C for 10 min to achieve proper labeling. Following, cells were washed by addition of buffer, collected by centrifugation and resuspended in 100#l of buffer that contained secondary 215 antibodies (AlexaFluor-488/594/647) and/or DAPI, diluted according to the manufacturer's specifications. After incubation for 10 min at 4°C and washing, cells were resuspended in 500 #l PBS and analyzed by flow cytometry (Canto II) or fractioned by FACS using a FACSAria™III cell sorter (Becton&Dickinson, BD). FACSisolated cells were collected in cell culture medium and seeded on appropriate vessels following centrifugation. Data analysis was performed with FlowJo (Ver 10.7.1).
Flow cytometric analyses were performed with FACS Canto II and sorting was performed using a FACS Aria III Data analysis was performed with FlowJo (Ver 10.7.1).

Reporter cells carrying NGFR and Ecad reporters were enriched
The population was defined by Forward (FSC)-and Side-Scatter (SSC) characteristics, excluding low sized cell debris and apoptotic cells (DAPI positive cells). Following, unstained control cells were investigated for adjustment of laser power and chanels for subsequenc analysis of stained cells. A figure exemplifying the gating strategy is provided in Supplementary figure 6d.
Female CD-1 nude mice (8-9 weeks of age, 24-26g, Charles River Laboratories) were stereotactically inoculated with 2.5E+04 BMC1-M1 and BMC1-M4 cells using a 1#l Hamilton syringe and a stereotactic frame as described previously. Briefly, the bur hole was placed 2 mm lateral (right) and 1 mm rostral from the bregma. The cells were administered at a depth of 3 mm. The number of cells used for the inoculation was determined in accordance with previous literature with the established human melanoma cell line M14.